JP2021518746A - Bacillus aryabhattai with silicon decomposition function and its use - Google Patents

Abstract

The present disclosure relates to the field of microorganisms, and specifically to Bacillus aryabhattai having a silicon decomposition function and its use. The strain is 1) deposited at the Ordinary Microbial Center of the China Microbial Species Preservation Management Committee, with a consignment number of CGMCC No: 17204 and a receipt date of January 16, 2019, and / or 2) Chinese microbial strain. It is selected from strains that have been deposited at the Ordinary Microbial Center of the Species Storage Management Committee, have a deposit number of CGMCC No: 17203, and have a receipt date of January 16, 2019. The strain has silicon decomposing ability, can promote plant growth, has alkali resistance, salt resistance, and anoxic resistance, and has a good application foreground. [Selection diagram] Fig. 3

Description

Cross-reference of related applications This application was filed with the China Patent Office on March 04, 2019, the application number is 201910161378.2, and the invention name is "Bacillus aryabhattai having a silicon decomposition function and its use" in China. Based on the patent application, priority is claimed, and the entire contents are incorporated in the present application by citation.

Technical Field The present application relates to the field of microorganisms, and specifically to Bacillus aryabhattai having a silicon decomposition function and its use.

Silicon is recognized by the international soil academia as the fourth nutrient element after nitrogen, phosphorus and potassium, and is a nutrient necessary for the growth of gramineous and root vegetable crops. Studies have shown that the addition of silicon improves the nitrogen content of ears in paddy rice, promotes the synthesis of starch and protein, and enriches the fruit of paddy rice, and silicon remarkably absorbs and accumulates nitrogen in winter melon. There is a positive correlation with the amount of silicon added in both the content of nitrogen and mineral elements during the maturation period. Increased content, which significantly or slightly improves potassium in the plant, the use of silicon fertilizers, the growth and production and quality of crops such as paddy rice, swallow, wheat, sugar cane, morokoshi It was revealed that it has the effect of improving. In addition to functioning in plant growth, silicon can also improve plant disease resistance and insect resistance, reduce poisoning and salt stress caused by metal ions, and improve drought resistance. The use of silicon fertilizers is crucial and practical, as predatory cultivation methods significantly reduce the content of effective silicon in the soil, and silicon-deficient cultivated land occupies more than 50% of the cultivated land area nationwide. It has significance.

In nature, silicon is slightly the second most widely distributed after oxygen and has the second highest content in the crust, predominantly as silicon dioxide and silicates. In the soil pot, the silicon content is about 70%, both of which are in a very stable crystalline and amorphous state, and their solubility is considerably low so that they are not easily absorbed by plants. Therefore, the selection of microorganisms capable of decomposing insoluble silicon-containing minerals in soil and the development of biosilicon fertilizers have become important means for solving problems related to silicon fertilizers. Microbial fertilizers can not only efficiently utilize insoluble silicon-containing minerals in soil, but also control diseases and improve soil, and can generate multiple benefits at once.

In China, silicate bacteria, such as Bacillus mucilaginosus, are often mentioned as silicon-degrading microorganisms. This is a bacterium capable of liberating potassium from water-insoluble potassium ore, and is also called a potassium bacterium. From this bacterium, we have developed a microbial fertilizer that enhances the usability of potash fertilizer.

Some reports have revealed that such bacteria have a silicon-degrading function. In addition, in foreign countries, it was reported that microorganisms such as pseudomonas, lysovium, and bacillus have a silicon-degrading function, but the ability of these bacteria to hydrolyze silicate minerals is not so strong, and they contain silicon. There is no report of developing a material that converts minerals into silicon fertilizer that is absorbed and used by plants.

In April 2009, Indian scientists discovered three new microbial bacteria in the upper stratosphere of the Earth. They do not exist on the ground, but have the ability to resist intense UV radiation. Scientists have found a total of 12 bacteria and a total of 6 fungi in the sample. Here, based on the 16S rRNA gene sequence, eight of them were found to have 98% similarity to known microbial species on the ground, while three of them were new. It was found to have much higher UV resistance than biologically close species. However, in current research, scientists have not yet elucidated the origins of the three microorganisms derived from this stratosphere. In future research, scientists will explore further to unravel the mystery of its origin. The first new microbial bacterium was named "Janibacter hoylei" after the respected astrophysicist Fred Hoyle. The bacterium of the second microorganism was named Bacillus isronensis, which indicates the success of the upcoming balloon experiment conducted by the Indian Space Research Organization (ISRO). The bacterium of the third microorganism was named "Aryabhata" after the famous Indian astronomer Aryabhata.

Traditionally, the Bacillus aryabhattai strain has been able to degrade bagasse (verified by a single strain) to produce glucose and fructose, with round colonies, yellowish rims, and smooth and regular edges. Was reported. However, there is not enough research on Bacillus aryabhattai, and in particular, there are few selections and studies of Bacillus aryabhattai strains having special functions.

The present disclosure relates to an isolated Bacillus aryabhattai strain selected from the following.
1) A strain that has been deposited at the China General Microbiological Culture Center (CGMCC) of the China Microbial Species Preservation Management Committee, with a consignment number of CGMCC No: 17204 and a receipt date of January 16, 2019.
And / or 2) A strain deposited at the Ordinary Microbial Center of the Chinese Microbial Species Preservation Management Committee, with a consignment number of CGMCC No: 17203 and a receipt date of January 16, 2019.

Both CGMCC No: 17203 and CGMCC No: 17204 have been deposited at the Ordinary Microbial Center of the China Microbial Species Conservation Management Committee, and the storage addresses are both No. 3 of Hokushin West Road, Chaoyang District, Beijing.

The disclosure further claims protection of progeny, mutants or derivatives of the strain.

In another aspect of the present disclosure, the present disclosure relates to a composition comprising a strain as described above.

In another aspect of the present disclosure, the present disclosure relates to the use of a strain as described above or a composition as described above in silicon degradation.

In another aspect of the present disclosure, the present disclosure further relates to a method of improving the health, growth or production of a plant, wherein an effective amount of the above composition is applied to the plant or the periphery of the plant. Including administration to the environment.

The present inventor unexpectedly discovered that the strain has a silicon-degrading ability. In addition, the strain can promote plant growth, has alkali resistance, salt resistance, and anoxic resistance, and has a good application foreground.

The present disclosure provides Bacillus aryabhattai strains selected from the following.
1) An isolated Bacillus aryabhattai strain I deposited at the Ordinary Microbial Center of the Chinese Microbial Species Preservation Management Committee, with a consignment number of CGMCC No: 17204 and a receipt date of January 16, 2019.
2) An isolated Bacillus aryabhattai strain II deposited at the Ordinary Microbial Center of the China Microbial Species Preservation Management Committee, with a deposit number of CGMCC No: 17203 and a receipt date of January 16, 2019.
3) A progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I having a silicon-degrading function.
And / or 4) Progeny, mutant or derivative of isolated Bacillus aryabhattai strain II having a silicon-degrading function.

In one or more embodiments, the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I, and the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain II further have a plant growth-promoting function. Has.

In one or more embodiments, the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I, and the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain II, further comprises the root dry matter weight of the crop. Has the function of increasing.

In one or more embodiments, the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I and the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain II contain 5% or less NaCl. It also has a silicon decomposition function in the culture system.

In one or more embodiments, the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I, and the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain II comprises 20% or less KNO3. It can grow in a culture system and also has a silicon decomposition function in a culture system containing 10% or less of KNO _3.

In one or more embodiments, the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I and the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain II are 6.5 ≤ pH ≤ 10. It also has a silicon decomposition function in the culture system of.

In one or more embodiments, the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I and the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain II are cultured at an oxygen concentration of 10% or higher. The system also has a silicon decomposition function.

In one or more embodiments, the 16S rRNA sequence of the isolated Bacillus aryabhattai strain I progeny, mutant or derivative has at least 99% identity with the 16S rRNA sequence of the isolated Bacillus aryabhattai strain I. Have.

In one or more embodiments, the 16S rRNA sequences of the isolated Bacillus aryabhattai strain II progeny, mutant or derivative have at least 99% identity with the 16S rRNA sequence of the isolated Bacillus aryabhattai strain II. Have.

The genomic sequence has at least 99% identity with the genomic sequence of the isolated Bacillus aryabhattai strain I.

In one or more embodiments, the genomic sequence of a progeny, mutant or derivative of the isolated Bacillus aryabhattai strain II has at least 90% identity with the genomic sequence of the isolated Bacillus aryabhattai strain II.

The present disclosure describes the isolated Bacillus aryabhattai strain I, the isolated Bacillus aryabhattai strain II, the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I, and / or the isolated Bacillus. A composition comprising a culture of progeny, mutant or derivative of aryabhattai strain II is provided.

In one or more embodiments, the composition is in the form of a liquid, frozen or dry powder.

In one or more embodiments, the composition is further selected from nutrients, fertilizers, acaricides, fungicides, fungicides, insecticides, microbiological agents, nematode insecticides, insecticides. Contains the compound or composition in an agriculturally effective amount.

In one or more embodiments, the composition further comprises a silicon fertilizer, which comprises a simple substance of silicon, an organosilicon compound and / or an inorganic silicon compound.

In one or more embodiments, the composition further comprises a potassium fertilizer, which comprises a simple metal of potassium, an organic potassium compound and / or an inorganic potassium compound.

The present disclosure provides the use of the Bacillus aryabhattai strain or composition described herein in silicon degradation.

In one or more embodiments, the silicon exists as a simple substance of silicon or as a silicon ion.

In one or more embodiments, the silicon exists as an aggregate of silicon ions and other elements, or as a bond complex with other elements.

In one or more embodiments, the silicon is present as a silicate or silicon-containing mineral.

In one or more embodiments, the silicon is present as a silicate.

In one or more embodiments, the silicate is selected from the group consisting of magnesium silicate, magnesium fluorosilicate, magnesium aluminum silicate, sodium silicate, potassium silicate and sodium potassium silicate. ..

In one or more embodiments, the silicon is present as a silicon-containing mineral.

In one or more embodiments, the silicon-containing minerals are nesosilicate minerals, solo silicate minerals, cyclosilicate minerals, inosilicate minerals, single-chain inosilicate minerals, double-chain inosilicate minerals, It is selected from the group consisting of phyllosilicate minerals and tect silicate minerals.

In one or more embodiments, the silicon exists as pyroxene, olivine, epidote, tourmaline, amphibole, mica, white clay, feldspar or quartz.

The present disclosure relates to a method of improving plant health, growth or production, said method comprising administering an effective amount of the composition described herein to the plant or the environment around the plant.

In one or more embodiments, the method of improving the health, growth or production of the plant comprises promoting root growth of the plant.

In one or more embodiments, the plant comprises wheat, paddy rice, corn, baby cabbage, tomatoes, cucumbers, broccoli, strawberries and / or grapes.

Hereinafter, in order to further clarify the specific embodiment or the technical means of the prior art of the present disclosure, the drawings necessary for describing the specific embodiment or the technical means of the prior art will be briefly described. As will be appreciated, the drawings described below are an embodiment of the present disclosure, and those skilled in the art will obtain other drawings based on these drawings, provided that they do not spend creative labor. be able to.

It is a figure which shows the measurement result of the silicon decomposition ability of the strain MB22 and the strain MB35-5 which concerns on one Embodiment of this disclosure. It is a figure which shows the measurement result of the salt resistance and the acid resistance / alkali resistance of the strain provided in one Embodiment of this disclosure. It is a figure which shows the silicon decomposition ability of the strain provided in one Embodiment of this disclosure under different culture conditions.

The Bacillus aryabhattai provided in the present disclosure
1) Strain with name MB35-5 and accession number CGMCC No: 17204, and 2) strain with name MB22 and accession number CGMCC No: 17203.
Both were deposited at the Ordinary Microbial Center of the Chinese Microbial Species Storage Management Committee, and were confirmed to be alive by the Storage Center on January 16, 2019.

Hereinafter, in order to further clarify the purpose, technical means and advantages of the embodiments of the present disclosure, the technical means according to the embodiments of the present disclosure will be clearly and completely described. In the embodiment, if the specific conditions are not specified, the normal conditions or the conditions recommended by the manufacturer shall be applied. As for the reagents or devices used, those for which the manufacturer is not specified shall be ordinary ones that can be purchased commercially.

Unless otherwise defined, the scientific and technical terms used herein should have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Hereinafter, exemplary methods and materials will be described, but methods and materials similar or identical to those described herein may also be used in the practice or testing of the present disclosure.

The present disclosure relates to an isolated Bacillus aryabhattai strain selected from the following.
1) A strain deposited at the Ordinary Microbial Center of the Chinese Microbial Species Preservation Management Committee, with a consignment number of CGMCC No: 17204 and a receipt date of January 16, 2019.
And / or 2) A strain deposited at the Ordinary Microbial Center of the Chinese Microbial Species Preservation Management Committee, with a consignment number of CGMCC No: 17203 and a receipt date of January 16, 2019.

The strain has a silicon decomposition function.
The silicon decomposition function is measured by the method specified in "3. Measurement of silicon decomposition ability" described in the examples of the present disclosure. The size of the silicon decomposition circle of the MB35-5 strain is about 4.5 mm, for example, 6 mm, 5 mm, 4 mm, 3.5 mm, 3 mm, 2.5 mm, and 2 mm.
The size of the silicon decomposition circle of the MB22 strain is about 6 mm, for example, 8 mm, 7 mm, 6.5 mm, 6 mm, 3 mm, 2.5 mm, and 2 mm.

In one aspect of the present disclosure, the present disclosure relates to a Bacillus aryabhattai strain having the following characteristics.
a) Being a descendant, mutant or derivative of the above strain, and b) having a silicon-degrading function.

The present disclosure claims protection of the Bacillus aryabhattai strain corresponding to the accession number, and a moderately mutated progeny, mutant or derivative strain with a fairly strong ability to degrade silicon.

As used herein, the technical term "descendant, mutant or derivative strain of Bacillus aryabhattai" refers to a Bacillus aryabhattai strain whose genome is highly similar to that of the MB35-5 or MB22 strain. Point to. In the present application, the mutant of the strain has an identity of 16S rRNA shown in SEQ ID NO: 1 (MB35-5) or SEQ ID NO: 2 (MB22) ≥99% (for example, 99.1% identity). , 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%). In one or more embodiments, the "Bacillus aryabahattai strain progeny, mutant or derivative strain" has at least 99%, 99.1% identity with the 16S rRNA of the MB35-5 or MB22 strain. It is 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.

The technical term "progeny, mutant or derivative strain of Bacillus aryabhattai strain" or mutant of strain can be curled because of its high degree of similarity in the genome. The genome of the Bacillus aryabhattai strain has at most 150 mutations, eg, 140, 130, 120, 110, 100, 90, 80, relative to the genome of the MB35-5 or MB22 strain. Includes 70, 60, 50, 40, 30 or 20 mutation events. Mutation events are limited as SNPs (single nucleotide polymorphisms) or INDELs (insertions, deletions, and combinations thereof). The number of mutation events is determined as follows. Using the genome of the MB35-5 strain as a control, mutation events existing in the genome of the mutant strain are detected, and each mutation event (SNP or INDEL) contains one mutation event (that is, for example, a small amount of nucleotides). Inserting a sequence to do so is considered as only one mutation event). In this context, the genomic sequences of the mutant strains of the present disclosure are limited by the number of mutational events contained in the MB35-5 or MB22 strain. Except for the quantity of mutation events, it is extra limited by the percentage of identity with the genomic sequence of the MB35-5 or MB22 strain. Here, in the present specification, the identity percentage indicates the percentage of a sequence found in the genome of one strain and existing in the genome of another strain. Specifically, a) a percentage of the sequences present in the genome of the mutant of the strain found in the genome of the MB35-5 or MB22 strain, or b) MB35 found in the genome sequence of the mutant of the strain. Shows the percentage of sequences present in the genome of -5 or MB22 strains. Therefore, the only difference from the given MB35-5 or MB22 strain is the mutant of the inserted (one or more) or deleted (one or more) strain, and the genome it has and the MB35-5 or MB22 strain. The identity percentage with the genome of one strain is 100%, because all the genome sequences of another strain are completely found in the genome of one strain. In one specific example, the genomic sequences of the mutant strains of the present disclosure, limited by the number of mutation events, have at least 90% identity percentage with the genomic sequences of the MB35-5 or MB22 strains. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5 %, 99.6%, 99.7%, 99.8%, 99.9%, 99.92%, 99.94%, 99.96%, 99.98% or 99.99%, and here. So, the identity percentage indicates the percentage of the sequence found in the genome of one strain and present in the genome of another strain. Identity is explained by comparing two genomic sequences over their full length (global alignment) and is calculated using one of the programs based on the Needleman-Wunsch algorithm.

CGMCC No: 17204 and / or CGMCC No: 17203 strains and their progeny, mutant or derivative strains have a plant growth-promoting effect, for example, promote plant root growth and increase crop root dry weight. increase.

CGMCC No: 17204 and / or CGMCC No: 17203 strain and its progeny, mutant or derivative strain have a silicon decomposition function even in a culture system containing 5% or less of NaCl.

In one or more embodiments, the concentration may be selected from, for example, 4.5% NaCl, 4% NaCl, 3.5% NaCl, 3% NaCl, 2.5% NaCl.
In one or more embodiments, the concentration may be selected from, for example, 4.5% NaCl or less, 4% NaCl or less, 3.5% NaCl or less, 3% NaCl or less, 2.5% NaCl or less. ..

CGMCC No: 17204 and / or CGMCC No: 17203 strain can grow in a culture system containing _{20% or less of KNO 3} and also has a silicon decomposition function in a culture system containing _{10% or less of KNO 3.} Have.
In one or more embodiments, the KNO ₃ concentration in the growth system is, for example, 19% KNO ₃ , 18% KNO ₃ , 17% KNO ₃ , 16% KNO ₃ , 15% KNO ₃ , 14% KNO ₃ , 13 % KNO ₃ , 12% KNO ₃ , 11% KNO ₃ , 10% KNO ₃ , 9% KNO ₃ , 8% KNO ₃ , 7% KNO ₃ , 6% KNO ₃ , 5% KNO ₃ , 4% KNO ₃ , 3 It may be selected from% KNO ₃ and 2% KNO _3.
In one or more embodiments, the KNO ₃ concentration in the growth system is, for example, 19% KNO ₃ or less, 18% KNO ₃ or less, 17% KNO ₃ or less, 16% KNO ₃ or less, 15% KNO ₃ or less, 14 % KNO ₃ or less, 13% KNO ₃ or less, 12% KNO ₃ or less, 11% KNO ₃ or less, 10% KNO ₃ or less, 9% KNO ₃ or less, 8% KNO ₃ or less, 7% KNO ₃ or less, 6% KNO _It may be selected from 3 or less, 5% KNO ₃ or less, 4% KNO ₃ or less, 3% KNO ₃ or less, and 2% KNO _{3 or less.}

In one or more embodiments, the KNO ₃ concentration in the silicon decomposition system is, for example, 9% KNO ₃ , 8% KNO ₃ , 7% KNO ₃ , 6% KNO ₃ , 5% KNO ₃ , 4% KNO ₃ , It may be selected from 3% KNO ₃ and 2% KNO _3.
In one or more embodiments, the KNO ₃ concentration in the silicon decomposition system is, for example, 9% KNO ₃ or less, 8% KNO ₃ or less, 7% KNO ₃ or less, 6% KNO ₃ or less, 5% KNO ₃ or less, It may be selected from 4% KNO ₃ or less, 3% KNO ₃ or less, and 2% KNO _{3 or less.}

CGMCC No: 17204 and / or CGMCC No: 17203 strain and its progeny, mutant or derivative strain also have a silicon decomposition function in a culture system of 6.5 ≤ pH ≤ 10.
The pH may be selected from 5, 6, 6.5, 7, 8, 9, and 10.

CGMCC No: 17204 and / or CGMCC No: 17203 strain and its progeny, mutant or derivative strain have a silicon decomposition function even in a culture system having an oxygen concentration of 10% or more.
In one or more embodiments, the oxygen concentration may be further selected from 15%, 20%, 25% or more.
In one or more embodiments, the oxygen concentration may be further selected from 15% or higher, 20% or higher, 25% or higher or higher.

The present disclosure further relates to a composition comprising a culture of the strain as described above.

In consideration of the fact that transportation may be required when actually applying the strain, the range of application of the Bacillus aryabhattai strain is expanded as a composition (particularly, a microbial agent) by an expansion culture method.

The compositions of the present disclosure (eg, when used as fermentant cultures) include, but are not limited to, simple cultures or mixed cultures.
Therefore, in the present disclosure, the simple culture is limited to that the whole or almost the whole of the culture consists of the same Bacillus aryabhattai strain of the present disclosure. In an alternative embodiment, the mixed culture is limited to a culture containing some microorganisms, specifically some bacterial strains, and the Bacillus aryabhattai strains and / or their progeny, mutants or derivative strains of the present disclosure. NS.

In some embodiments, the composition further comprises a strain capable of degrading potassium.

The composition is used in agriculture and may be in the form of a liquid, frozen or dry powder, or is often used in the art such as granules, suspensions, wettable powders, emulsions or liquids. Represented in the form of the formulation used.

The carrier may be any biologically inert carrier, which is often used for agricultural chemicals in agriculture and horticulture, and can be used in any solid or liquid form. It is not limited to any particular carrier.

In some specific embodiments, the composition, in the form of a frozen or dry powder, comprises a carrier in a solid state.

Carriers in the solid state are, for example, high in clay, talc, bentonite, zeolite, calcium carbonate, diatomaceous earth and ore flour such as white carbon, plant milling such as corn flour and starch, and polyalkylene glycol such as polyvinyl alcohol. Contains molecular compounds. On the other hand, typical liquid carriers include various organic solvents such as decane and decane, vegetable oils and mineral oils, and water.

In some embodiments, the solid carrier is pyrophyllite, turf, talc, subcarbonate, pyrophyllite, montmorillonite, alginate, pressed dehydrated sludge, shavings, pearlite, mica, silica, quartz powder, calcium bentonite, Contains one or more of vermiculite, kaolin, light calcium carbonate, diatomaceous earth, barley stone, bentonite, zeolite, white carbon black, fine sand and clay.

In some embodiments, the composition comprises an auxiliary agent. For example, the composition usually includes, but is not limited to, surfactants, pressure-sensitive adhesives, stabilizers and the like used as auxiliaries in agricultural and horticultural chemical products, which may be used alone or as required. And / or stabilizers, such as antioxidants and / or pH regulators. In some cases, light stabilizers can be used.

The total amount of these auxiliaries may be 0 wt% to 80 wt%, and the content of the carrier is a value obtained by subtracting the amount of the active ingredient and the content of the auxiliaries from 100 wt%.

In some specific embodiments, the auxiliaries are sodium dodecylbenzene sulfonate, sodium butylnaphthalene sulfonate, trehalose, glycerin, sodium lignin sulfonate, sodium alkylnaphthalene sulfonate polycondensate, niacin, alcohol, buffer. Contains one or more of salts, sodium chloride, amino acids, vitamins, proteins, polypeptides, polysaccharides or monosaccharides, yeast extracts, white carbon black, tea saponins, defatted milk.

In some specific embodiments, the composition is a compound selected from nutrients, fertilizers, acaricides, fungicides, fungicides, insecticides, microbial killers, nematodes, insecticides. Alternatively, the composition is contained in an agriculturally effective amount. For example, the composition further comprises fertilizers such as silicone fertilizers and potash fertilizers. Silicon fertilizer is in the form of elemental silicon, organosilicon compounds and / or inorganic silicon compounds. Potassium fertilizer is in the form of elemental potassium metals, organic potassium compounds and / or inorganic potassium compounds.

The above components can be combined with the strains provided by the present application to achieve better technical effects.

In some embodiments, in the composition, viable counts of the Bacillus Aryabhattai strain ^is 10 7~12 cfu · ^{mL -1} or ^{10 7~12} cfu · ^{g -1.}
Alternatively, it may be selected from ^{10 8} cfu ・ mL ^-1 , 10 ⁹ cfu ・ mL ^-1 , 10 ¹⁰ cfu ・ mL ^-1 , and 10 ¹¹ cfu ・ mL ^-1. Alternatively, it may be selected from ^{10 8} cfu ・ g ^-1 , 10 ⁹ cfu ・ g ^-1 , 10 ¹⁰ cfu ・ mL ^-1 , and 10 ¹¹ cfu ・ mL ^-1.

The present disclosure relates to the use of such strains or compositions as described above in silicon degradation.

In some embodiments, the silicon exists as a simple substance of silicon or as a silicon ion.

In some embodiments, the silicon exists as an aggregate of silicon ions and other elements, or as a bond complex with other elements.

In some embodiments, the silicon is present as a silicate or silicon-containing mineral.
In one or more embodiments, the silicate may be selected from magnesium silicate, magnesium fluorosilicate, magnesium aluminum silicate, sodium silicate, potassium silicate and sodium potassium silicate.
In one or more embodiments, the silicon-containing minerals are nesosilicate minerals, solo silicate minerals, cyclosilicate minerals, inosilicate minerals, single-chain inosilicate minerals, double-chain inosilicate minerals, phyllo. You may choose from silicate minerals and tect silicate minerals.

Common forms of silicon in one or more embodiments include, for example, pyroxene, olivine, epidote, tourmaline, amphibole, mica, white clay, feldspar or quartz.

The present disclosure relates to a method of improving plant health, growth or production, said method comprising administering an effective amount of the composition described herein to the plant or the environment around the plant.

Hereinafter, the embodiment of the present disclosure will be described in more detail by way of examples, but the following examples are for explaining the present invention and do not limit the present invention in any way. In the examples, if the specific conditions are not specified, the normal conditions or the conditions recommended by the manufacturer shall be applied. As for the reagents or devices used, those for which the manufacturer is not specified shall be ordinary ones that can be purchased commercially.

Experimental procedure

1 Selection of bacterial species 1.1 Soil sample collection Typical soils such as sand soil, clay, and black soil were selected as the collection sites. Samples may come from different areas such as upland fields, pastures, and forest soil. In particular, samples were taken from paddy rice / wheat fields and soils where silicon fertilizer was used for many years. A sample of 15 to 20 g was collected at each location, and the collection location (province, prefecture), collection date, and soil origin (plant, sand soil, or other) of the collected sample were specified. The sample was placed in a tube containing 80% glycerin and stored in a refrigerator at -80 ° C.

1.2 Enrichment of target microorganisms in soil sample Enrichment of step 1 1 g of soil sample was taken in 10 mL pure water and shaken to obtain a mixed solution. Mixed with nutrient-deficient R2A liquid medium (yeast powder 0.5 g, tryptone 0.5 g, glucose 0.5 g, dipotassium hydrogen phosphate 0.3 g, sodium pyruvate 0.3 g, magnesium silicate 5.0 g, water 1 L) 100 μL of the solution was taken and shake-cultured at 30 ° C. for 3 days to obtain a cultured bacterial solution. After diluting the cultured bacterial solution ^10-6 , ^10-7 , ^10-8 times, R2A solid medium (yeast powder 0.5 g, tryptone 0.5 g, peptone 0.75 g, glucose 0.5 g, soluble starch 0. 5 g, dipotassium hydrogen phosphate 0.3 g, sodium pyruvate 0.3 g, magnesium silicate 2.5 g, agar 15 g, water 1 L) were applied to select a strain having a function.

Enrichment of Step 2 Take 100 μL of the bacterial solution prepared using the strain obtained in Step 1 and take 1 / 2N nutrient-deficient R2A medium (yeast powder 0.25 g, tryptone 0.25 g, glucose 0.5 g, hydrogen phosphate). Bacterial solution was obtained by shaking culture at 30 ° C. for 3 days in 0.3 g of dipotassium, 0.3 g of sodium pyruvate, 5.0 g of magnesium silicate, and 1 L of water). The cultured bacterial solution was ^{diluted 10-6} , ^10-7 , ^10-8 times, and then applied to a petri dish to select a strain having a silicon decomposition circle function.

Enrichment of Step 3 100 μL of the bacterial solution prepared using the strain obtained in Step 2 was taken, and the bacterial solution was cultured in 1 / 2N nutrient-deficient R2A medium by shaking culture at 30 ° C. for 3 days to obtain a cultured bacterial solution. The cultured bacterial solution was ^{diluted 10-6} , ^10-7 , ^10-8 times, and then applied to a petri dish to select a strain having a silicon decomposition circle function.

1.3 Repeated verification In order to ensure the sustainability of the strain's ability to decompose silicon, the strain selected from the enriched petri dish was seeded in R2A solid medium containing 0.25% magnesium silicate and 30 ° C. Was cultured in. The experimental results were observed daily to verify whether the selected colonies had silicon function and to eliminate false positive colonies.

2 Bacterial species identification 2.1 Bacterial DNA extraction by CTAB method 1) A single colony was inoculated on 5 mL of R2A medium and cultured overnight at 30 ° C. to obtain a seed culture solution.
2) 1 mL of the seed culture solution was taken, seeded in 100 mL of R2A liquid medium, and cultured under the conditions of 37 ° C. and 220 r / min for 16 hours to obtain a culture.
3) The culture was centrifuged at 5000 r / min for 10 minutes, and the supernatant was removed to obtain bacterial cells. The cells were washed by centrifugation with 10 mL of TE, and then the cells were suspended with 10 mL of TE to obtain a suspension. After mixing uniformly, it was stored at −20 ° C.
4) 3.5 mL of the bacterial cell suspension was taken, 184 μL of 10% SDS was added and mixed uniformly, and 37 μL of 10 mg / mL proteinase K was added and mixed uniformly. Incubated at 37 ° C. for 1 hour.
5) 740 μL of 5 mol / L NaCl was added, and 512 μL of CTAB / NaCl was further added, mixed uniformly, and incubated at 65 ° C. for 10 minutes.
6) The same volume of chloroform / isoamyl alcohol was added, mixed uniformly, and centrifuged at 10000 r / min for 5 minutes to leave a supernatant.
7) The same volume of phenol: chloroform: isoamyl alcohol (25:24: 1) was added to the supernatant, mixed uniformly, and centrifuged at 10000 r / min for 5 minutes to leave the supernatant.
8) 0.6 times the amount of isopropanol was added to the supernatant, mixed uniformly, centrifuged at 10000 r / min for 5 minutes, DNA precipitates were collected, and the mixture was centrifuged with 70% alcohol and washed.
9) The DNA precipitate was dissolved in 1 mL of TE, RNase A having a final concentration of 20 μg / mL was added, and the mixture was stored at 4 ° C.

2.2 Amplification and sequence Using the extracted DNA as a template, 16S rDNA by PCR using 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-GGTTACCTTGTTACGACTT-3'), which are universal primers for 16S rDNA amplification. Amplified. The PCR reaction conditions were pre-denaturation at 94 ° C. for 30 seconds, denaturation at 94 ° C. for 30 seconds, annealing at 52 ° C. for 30 seconds, and extension at 72 ° C. for 60 seconds in 35 cycles. The PCR product was subjected to 1.5% agarose gel electrophoresis. The PCR product was subjected to agarose gel electrophoresis, recovered, purified, and then sequenced (Beijing Bibing Biological Technology Co., Ltd.). Based on the obtained 16S rDNA sequence, a homologous sequence was searched by BLAST (Basic Local Element Search Tool) in GenBank. In addition, a phylogenetic tree was created using MEGA 5.0 software.

3 Measurement of silicon decomposition ability The strain selected in the previous step was inoculated on R2A medium containing 0.5% magnesium silicate. Silicate bacteria were used as positive controls. Forty-eight hours after sowing, the colonies were washed away, the diameter of the silicon decomposition circle in the medium was measured, and the unit was mm.
Silicon decomposition capacity = diameter of silicon decomposition circle (mm) + X
Here, X is a weighting coefficient, and is set to -2, -1, 0, 1, and 2, respectively, depending on the transparency of the silicon decomposition circle of the strain.

4 Observation of strain morphology After seeding the selected strain on an R2A plate and culturing at 30 ° C. for 24 hours, the size, shape, color, glossiness, adhesiveness, ridge shape, transparency, edge characteristics and margins The presence or absence of spores was observed.

5 strains measured sorted strain stress tolerance, pH 5, were seeded into R2A medium _{pH10,5% NaCl, 5% KNO 3} , 10% KNO 3, 20% KNO 3, whether strains to grow, also Whether the growth was suppressed and the rate of growth were observed. Furthermore, the silicon decomposition ability of the strain in R2A medium (containing 1.0% magnesium silicate) of pH 5, pH 10, 5% NaCl, 10% NaCl, 5% KNO ₃ , 10% KNO _{3 was measured and 3} .. It was calculated by the formula described in.

6 Measurement of aerobicity of strains The oxygen concentration was controlled to 15% and 10% using carbon dioxide and aneropack for microaerobic culture manufactured by Mitsubishi Gas Chemicals, Ltd., and the growth status of the strains under oxygen-deficient conditions. And the silicon decomposition ability was verified.

7 Hemolyticity test The bacterial species selected above were blood agar plate medium (Pancreatic digest of casein) 10.0 g, heart pancreatin digestion product 3.0 g, corn starch 1.0 g, The mixture was seeded on 13.0 g of agar, 5.0 g of digested product of meat with pepsin, 5.0 g of yeast extract, 5.0 g of sodium chloride, 1000 mL of distilled water, and 60 mL of sheep blood), and cultured in a constant temperature box at 30 ° C. for 18 hours. The morphology of the colonies was observed, and whether or not a hemolytic circle appeared was observed.

8 Measurement of spore formation rate In aseptic operation, clean slides were flame sterilized with alcohol, the area of the specimen was clearly marked, and a drop of phosphate buffer was added into the area (0.2 M, pH 7.2). ). Two or three colonies were taken with a seeding loop, uniformly applied to the dropped phosphate buffer or one drop of the bacterial cell suspension so as to form a thin film, and dried and fixed. After drying, a 7.6% malachite green solution was added dropwise to the cell membrane so as to cover the entire area of the applied bacterial cell membrane, and the cells were stained for 15 to 20 minutes, and the flowing water was green. It was washed with water and decolorized until it completely disappeared. After straining and drying the surface water, re-stain, add 0.5% safranin solution and dye for 2-3 minutes, turn over to remove the dye, and rinse the dye with water a little. The residual liquid was sucked up with a filter paper, dried, and then used as a spore colony specimen. After drying, when observed with an oil-immersion lens, the spores showed green color, and the spore sac and vegetative body showed red color.

At least three visual fields were observed, the number of spores and the number of bacteria in each visual field were counted, and the average value (A) of the number of spores and the average value (B) of the number of bacteria were recorded and calculated. The spore formation rate by microscopic examination was calculated by the following formula.
Spore formation rate by microscopic examination: N = A / (A + B) × 100%
In the formula, N represents the spore formation rate by microscopic examination of the sample, A represents the average value of the number of spores in the microscopic field of view of the sample, and B represents the average value of the number of bacteria in the microscopic field of view of the sample. Is.

9 Pot test Using vermiculite as a substrate, 500 g of soil was added to each pot, and the mixture was moistened with an equal amount and a small amount of water. Seeds of "Chung Mono 958" and popcorn (Otani Prefectural Green Treasure Seed Industry Co., Ltd.) of uniform size were selected, 6 seeds were sown in 1 pot, and 4 pots were repeated. After diluting the bacterial solution 50 times, an equal amount of the bacterial solution was poured in an appropriate amount in a timely manner according to the demand for corn growth. After 15 days, the root dry matter was weighed. The nutrient-deficient R2A liquid medium in which no strain was seeded was used as the blank control CK1, and the medium in which the silicate bacteria were seeded was used as the positive control CK2.

Experimental result

1 Strain selection This test was conducted on 34 soils of 19 crops including wheat, paddy rice, corn, baby cabbage, tomatoes, cucumbers, broccoli, strawberries and / or grapes from a total of 10 provinces and 16 cities / prefectures. A sample was taken. A total of 206 strains having a silicon-degrading function were selected and obtained by enrichment experiments. As verified by 16S rDNA sequence, silicon decomposition ability, stress resistance, aerobicity, hemolysis, strain growth characteristics, spore formation rate, and pot test, it has biological safety, high spore formation rate, and silicon decomposition. Gram-positive bacteria-MB22 and MB35-5, which have strong ability, resistance to alkali and salt, can grow well under low oxygen conditions, do not generate hemolytic circles, and have a plant growth promoting effect, were selected. From the results of analyzing the 16S rDNA sequences of these two strains, it is clear that the identity between them and Bacillus aryabhattai is as high as 99%.

2 Strain morphology When grown in R2A medium for 2 days, the colonies were round, beige and opaque, the surface was smooth and moist, the edges were regular, and halos were observed. It has a protrusion in the center, 1.5 to 2 mm in diameter, is circular, glossy but sticky, has slight bumps, and has smooth and regular edges. ..

3 Silicon decomposition ability From Table 1 and FIG. 1, it is clear that the silicon decomposition ability of the strains MB22 and MB35-5 is significantly higher than that of the control silicate strain 3016.

4 Stress resistance of strains From the measurement results shown in Table 2 and FIG. 2, the strains MB22 and MB35-5 according to the present disclosure have superior salt and alkali resistance as compared with the control silicate strain 3016, and It has been shown to have a certain degree of acid resistance.

5 From the measurement results of silicon decomposition ability under stress conditions (Table 3), the strain according to the present disclosure still has a silicon decomposition function under high salt concentration conditions, strong alkaline conditions and weakly acidic conditions, while a control strain. It was revealed that the silicate bacterium 3016 does not grow well under the stress conditions of any of the tests.

6 Survival and silicon decomposition ability of strains under oxygen-deficient conditions From the results in Table 4, the strains MB22 and MB35-5 according to the present disclosure are the control silicate strains and the control silicate strains under both oxygen-deficient and non-oxygen-deficient conditions. It has been shown to be significantly superior to other bacterial strains.

7 Hemolytic test Neither of the two strains provided by this disclosure has a hemolytic circle. From the experiments, it has been clarified that the hemolytic reaction does not occur in the strain according to the present disclosure.

8 Measurement of spore formation rate From the results shown in Table 5, the spore formation rate of the strains MB22 and MB35-5 according to the present disclosure was significantly higher than that of the control strain silicate strain 3016 under the same conditions in this experiment. Has been clarified.

9 Pot Test From the results shown in Table 6, the silicate bacteria 3016 and the strains MB22 and MB35-5 according to the present disclosure have an action of promoting root growth of corn, but the action of promoting the strains MB22 and MB35-5 is silicic acid. It has been shown to be significantly superior to the salt bacteria and the other two strains used in the experiment, MB15-13 and MB12-7. The two strains according to the present disclosure have a root growth promoting effect on wheat, but not as much as corn. The control silicate bacterium 3016 has no root growth promoting effect on wheat. The other two strains are effective for wheat development and growth.

It should be noted that the above examples are used only for explaining the present invention, and are not for limiting the present invention. Although the present disclosure has been described in detail with reference to the above embodiment, those skilled in the art to which this disclosure belongs should understand that the technical scheme described in the above embodiment can be modified. Alternatively, some or all of the technical features can be replaced with equivalents. It will be understood that the essence of the technical plan according to the content of the modification or replacement does not deviate from the scope of the technical plan of each embodiment according to the present disclosure.

The strain according to the present disclosure has a silicon decomposing ability, can promote plant growth, has alkali resistance, salt resistance, and anoxic resistance, and has a good application foreground. The strain can be used to improve plant health, growth or production.

Claims (20)

Bacillus aryabhattai strain selected from the following.
1) An isolated Bacillus aryabhattai strain I deposited at the Ordinary Microbial Center of the Chinese Microbial Species Preservation Management Committee, with a consignment number of CGMCC No: 17204 and a receipt date of January 16, 2019.
2) An isolated Bacillus aryabhattai strain II deposited at the Ordinary Microbial Center of the China Microbial Species Preservation Management Committee, with a deposit number of CGMCC No: 17203 and a receipt date of January 16, 2019.
3) A progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I having a silicon-degrading function.
And / or 4) Progeny, mutant or derivative of isolated Bacillus aryabhattai strain II having a silicon-degrading function. The progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I, and the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain II have a plant growth promoting function, preferably root dry matter weight of the crop. The Bacillus aryabhattai strain according to claim 1, which has a function of increasing the amount of strain. The progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I, and the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain II have a silicon-degrading function even in a culture system containing 5% or less of NaCl. , The Bacillus aryabhattai strain according to claim 1 or 2. The progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I and the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain II can grow in a culture system containing 20% or less of KNO3. The Bacillus aryabhattai strain according to any one of claims 1 to 3, which also has a silicon decomposition function even in a culture system containing 10% or less of KNO3. The progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I, and the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain II have a silicon-degrading function even in a culture system of 6.5 ≤ pH ≤ 10. The Bacillus aryabhattai strain according to any one of claims 1 to 4. The progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I, and the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain II have a silicon decomposition function even in a culture system having an oxygen concentration of 10% or more. The Bacillus aryabhattai strain according to any one of claims 1 to 5. Any of claims 1-6, wherein the 16S rRNA sequence of the isolated Bacillus aryabhattai strain I progeny, mutant or derivative has at least 99% identity with the isolated Bacillus aryabhattai strain I 16S rRNA sequence. The Bacillus aryabhattai strain according to item 1. Any of claims 1-7, wherein the 16S rRNA sequence of a progeny, mutant or derivative of the isolated Bacillus aryabhattai strain II has at least 99% identity with the 16S rRNA sequence of the isolated Bacillus aryabhattai strain II. The Bacillus aryabhattai strain according to item 1. Any one of claims 1 to 6, wherein the genomic sequence of the isolated Bacillus aryabhattai strain I progeny, mutant or derivative has at least 90% identity with the isolated Bacillus aryabhattai strain I genomic sequence. The Bacillus aryabhattai strain according to the section. Any one of claims 1 to 7, wherein the genomic sequence of the isolated Bacillus aryabhattai strain II progeny, mutant or derivative has at least 90% identity with the isolated Bacillus aryabhattai strain II genomic sequence. The Bacillus aryabhattai strain according to the section. The isolated Bacillus aryabhattai strain I according to any one of claims 1 to 10, the isolated Bacillus aryabhattai strain II, the progeny, mutant or derivative of the isolated Bacillus aryabhattai strain I, and / or isolated. A composition comprising a culture of offspring, mutants or derivatives of Bacillus aryabhattai strain II.
Preferably, the composition is in the form of a liquid, frozen or dry powder.
Preferably, the composition further agriculturals a compound or composition selected from nutrients, fertilizers, acaricides, fungicides, fungicides, insecticides, microbiological agents, nematodes, insecticides. Composition containing in an effective amount for use. The composition according to claim 11, wherein the composition contains a silicon fertilizer, and the silicon fertilizer contains a simple substance of silicon, an organosilicon compound and / or an inorganic silicon compound. The composition according to claim 11 or 12, wherein the composition contains a potassium fertilizer, and the potassium fertilizer contains a simple substance metal of potassium, an organic potassium compound and / or an inorganic potassium compound. The use of the Bacillus aryabhattai strain according to any one of claims 1 to 10 or the composition according to claim 11 in silicon decomposition.
Preferably, the silicon exists as a simple substance of silicon or as a silicon ion.
Preferably, the silicon exists as an aggregate of silicon ions and other elements, or as a bond complex of other elements.
Preferably, the silicon is used as a silicate or a silicon-containing mineral. The silicon exists as a silicate and
Preferably, the silicate is selected from the group consisting of magnesium silicate, magnesium fluorosilicate, magnesium aluminum silicate, sodium silicate, potassium silicate and sodium potassium silicate, according to claim 14. Use of. The silicon exists as a silicon-containing mineral and
The silicon-containing minerals are preferably nesosilicate minerals, solo silicate minerals, cyclosilicate minerals, inosilicate minerals, single-chain inosilicate minerals, double-chain inosilicate minerals, phyllosilicate minerals, and tect. The use according to claim 14, which is selected from the group consisting of silicate minerals. The use according to claim 14, wherein the silicon exists as pyroxene, olivine, epidote, tourmaline, amphibole, mica, white clay, feldspar or quartz. A way to improve plant health, growth or production
A method comprising administering to the plant or the surrounding environment of the plant an effective amount of the composition according to any one of claims 11 to 13. 18. The method of claim 18, wherein the method of improving the health, growth or production of the plant comprises promoting root growth of the plant. The method of claim 18 or 19, wherein the plant comprises wheat, paddy rice, corn, baby cabbage, tomatoes, cucumbers, broccoli, strawberries and / or grapes.

Images